Quantification of mRNA using real-time RT-PCR

Abstract

The real-time reverse transcription polymerase chain reaction (RT-qPCR) addresses the evident requirement for quantitative data analysis in molecular medicine, biotechnology, microbiology and diagnostics and has become the method of choice for the quantification of mRNA. Although it is often described as a “gold” standard, it is far from being a standard assay. The significant problems caused by variability of RNA templates, assay designs and protocols, as well as inappropriate data normalization and inconsistent data analysis, are widely known but also widely disregarded. As a first step towards standardization, we describe a series of RT-qPCR protocols that illustrate the essential technical steps required to generate quantitative data that are reliable and reproducible. We would like to emphasize, however, that RT-qPCR data constitute only a snapshot of information regarding the quantity of a given transcript in a cell or tissue. Any assessment of the biological consequences of variable mRNA levels must include additional information regarding regulatory RNAs, protein levels and protein activity. The entire protocol described here, encompassing all stages from initial assay design to reliable qPCR data analysis, requires approximately 15 h.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Steps involved in planning a RT-qPCR assay.
Figure 2: RT-qPCR experimental workflow.
Figure 3: The 3′:5′ assay used to estimate mRNA integrity.
Figure 4: Optimization of primer concentration.
Figure 5: Acceptable and unacceptable standard curves.
Figure 6: Outline of reverse transcription protocol.
Figure 7: The relationship between amplification plot and standard curve.
Figure 8: Invalid Ct generated by spurious amplification plot.
Figure 9: Amplification plots demonstrating the importance of appropriately adjusting the baseline.
Figure 10: SYBR Green melt curve demonstrating the co-amplification of an mRNA target (lower Tm) and genomic DNA with a short intron.
Figure 11: SYBR Green melt curve demonstrating the appearance of primer dimers in a poorly designed assay.
Figure 12: Comparison of SYBR Green I and TaqMan amplification plots.

References

  1. 1

    Gibson, U.E., Heid, C.A. & Williams, P.M. A novel method for real time quantitative RT-PCR. Genome Res. 6, 995–1001 (1996).

    CAS  Article  Google Scholar 

  2. 2

    Bustin, S.A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J. Mol. Endocrinol. 25, 169–193 (2000).

    CAS  Article  Google Scholar 

  3. 3

    Ginzinger, D.G. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream. Exp. Hematol. 30, 503–512 (2002).

    CAS  Article  Google Scholar 

  4. 4

    Bustin, S.A. & Nolan, T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J. Biomol. Tech. 15, 155–66 (2004).

    PubMed  PubMed Central  Google Scholar 

  5. 5

    Stahlberg, A., Hakansson, J., Xian, X., Semb, H. & Kubista, M. Properties of the reverse transcription reaction in mRNA quantification. Clin. Chem. 50, 509–515 (2004).

    CAS  Article  Google Scholar 

  6. 6

    Stahlberg, A., Kubista, M. & Pfaffl, M. Comparison of reverse transcriptases in gene expression analysis. Clin. Chem. 50, 1678–1680 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Bustin, S.A. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29, 23–39 (2002).

    CAS  Article  Google Scholar 

  8. 8

    Bustin, S.A. & Mueller, R. Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis. Clin. Sci. (Lond.) 109, 365–379 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Bustin, S.A. & Mueller, R. Real-time reverse transcription PCR and the detection of occult disease in colorectal cancer. Mol. Aspects Med. 27, 192–223 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Higuchi, R., Dollinger, G., Walsh, P.S. & Griffith, R. Simultaneous amplification and detection of specific DNA sequences. Nat. Biotechnol. 10, 413–417 (1992).

    CAS  Article  Google Scholar 

  11. 11

    Higuchi, R., Fockler, C., Dollinger, G. & Watson, R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnol. (NY) 11, 1026–1030 (1993).

    CAS  Google Scholar 

  12. 12

    Bustin, S.A. Real-time, fluorescence-based quantitative PCR: a snapshot of current procedures and preferences. Expert Rev. Mol. Diagn. 5, 493–498 (2005).

    CAS  Article  Google Scholar 

  13. 13

    Bustin, S.A., Benes, V., Nolan, T. & Pfaffl, M.W. Quantitative real-time RT-PCR-a perspective. J. Mol. Endocrinol. 34, 597–601 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Bustin, S.A. A-Z of Quantitative PCR (IUL Press, La Jolla, California, 2004).

    Google Scholar 

  15. 15

    Fleige, S. & Pfaffl, M.W. RNA integrity and the effect on the real-time qRT-PCR performance. Mol. Aspects Med. 27, 126–139 (2006).

    CAS  Article  Google Scholar 

  16. 16

    Schroeder, A. et al. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol. Biol. 7, 3 (2006).

    Article  Google Scholar 

  17. 17

    Auer, H. et al. Chipping away at the chip bias: RNA degradation in microarray analysis. Nat. Genet. 35, 292–293 (2003).

    CAS  Article  Google Scholar 

  18. 18

    Radstrom, P., Knutsson, R., Wolffs, P., Lovenklev, M. & Lofstrom, C. Pre-PCR processing: strategies to generate PCR-compatible samples. Mol. Biotechnol. 26, 133–146 (2004).

    Article  Google Scholar 

  19. 19

    Lefevre, J., Hankins, C., Pourreaux, K., Voyer, H. & Coutlee, F. Prevalence of selective inhibition of HPV-16 DNA amplification in cervicovaginal lavages. J. Med. Virol. 72, 132–137 (2004).

    CAS  Article  Google Scholar 

  20. 20

    Sunen, E., Casas, N., Moreno, B. & Zigorraga, C. Comparison of two methods for the detection of hepatitis A virus in clam samples (Tapes spp.) by reverse transcription-nested PCR. Int. J. Food Microbiol. 91, 147–154 (2004).

    CAS  Article  Google Scholar 

  21. 21

    Perch-Nielsen, I.R., Bang, D.D., Poulsen, C.R., El-Ali, J. & Wolff, A. Removal of PCR inhibitors using dielectrophoresis as a selective filter in a microsystem. Lab Chip 3, 212–216 (2003).

    CAS  Article  Google Scholar 

  22. 22

    Jiang, J., Alderisio, K.A., Singh, A. & Xiao, L. Development of procedures for direct extraction of Cryptosporidium DNA from water concentrates and for relief of PCR inhibitors. Appl. Environ. Microbiol. 71, 1135–1141 (2005).

    CAS  Article  Google Scholar 

  23. 23

    Guy, R.A., Payment, P., Krull, U.J. & Horgen, P.A. Real-time PCR for quantification of Giardia and Cryptosporidium in environmental water samples and sewage. Appl. Environ. Microbiol. 69, 5178–5185 (2003).

    CAS  Article  Google Scholar 

  24. 24

    Stahlberg, A., Zoric, N., Aman, P. & Kubista, M. Quantitative real-time PCR for cancer detection: the lymphoma case. Expert Rev. Mol. Diagn. 5, 221–230 (2005).

    CAS  Article  Google Scholar 

  25. 25

    Stahlberg, A., Aman, P., Ridell, B., Mostad, P. & Kubista, M. Quantitative real-time PCR method for detection of B-lymphocyte monoclonality by comparison of kappa and lambda immunoglobulin light chain expression. Clin. Chem. 49, 51–59 (2003).

    CAS  Article  Google Scholar 

  26. 26

    Tichopad, A., Dilger, M., Schwarz, G. & Pfaffl, M.W. Standardized determination of real-time PCR efficiency from a single reaction set-up. Nucleic Acids Res. 31, e122 (2003).

    Article  Google Scholar 

  27. 27

    Ramakers, C., Ruijter, J.M., Deprez, R.H. & Moorman, A.F. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339, 62–66 (2003).

    CAS  Article  Google Scholar 

  28. 28

    Liu, W. & Saint, D.A. Validation of a quantitative method for real time PCR kinetics. Biochem. Biophys. Res. Commun. 294, 347–353 (2002).

    CAS  Article  Google Scholar 

  29. 29

    Pasloske, B.L., Walkerpeach, C.R., Obermoeller, R.D., Winkler, M. & DuBois, D.B. Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards. J. Clin. Microbiol. 36, 3590–3594 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Cloud, J.L. et al. Description of a multiplex Bordetella pertussis and Bordetella parapertussis LightCycler PCR assay with inhibition control. Diagn. Microbiol. Infect. Dis. 46, 189–195 (2003).

    CAS  Article  Google Scholar 

  31. 31

    Nolan, T., Hands, R.E., Ogunkolade, B.W. & Bustin, S.A. SPUD: a qPCR assay for the detection of inhibitors in nucleic acid preparations. Anal. Biochem. 351, 308–310 (2006).

    CAS  Article  Google Scholar 

  32. 32

    Cusi, M.G., Valassina, M. & Valensin, P.E. Comparison of M-MLV reverse transcriptase and Tth polymerase activity in RT-PCR of samples with low virus burden. Biotechniques 17, 1034–1036 (1994).

    CAS  PubMed  Google Scholar 

  33. 33

    Juhasz, A., Ravi, S. & O'Connell, C.D. Sensitivity of tyrosinase mRNA detection by RT-PCR: rTth DNA polymerase vs. MMLV-RT and AmpliTaq polymerase. Biotechniques 20, 592–600 (1996).

    CAS  PubMed  Google Scholar 

  34. 34

    Easton, L.A., Vilcek, S. & Nettleton, P.F. Evaluation of a 'one tube' reverse transcription-polymerase chain reaction for the detection of ruminant pestiviruses. J. Virol. Methods 50, 343–348 (1994).

    CAS  Article  Google Scholar 

  35. 35

    Vandesompele, J., De Paepe, A. & Speleman, F. Elimination of primer-dimer artifacts and genomic coamplification using a two-step SYBR green I real-time RT-PCR 303, 95–98 (2002).

  36. 36

    Mader, R.M. et al. Reverse transcriptase template switching during reverse transcriptase-polymerase chain reaction: artificial generation of deletions in ribonucleotide reductase mRNA 137, 422–428 (2001).

  37. 37

    Suslov, O. & Steindler, D.A. PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency. Nucleic Acids Res 33, e181 (2005).

    Article  Google Scholar 

  38. 38

    Lacey, H.A., Nolan, T., Greenwood, S.L., Glazier, J.D. & Sibley, C.P. Gestational profile of Na+/H+ exchanger and Cl-/HCO3- anion exchanger mRNA expression in placenta using real–time QPCR. Placenta 26, 93–98 (2005).

    CAS  Article  Google Scholar 

  39. 39

    Stangegaard, M., Dufva, I.H. & Dufva, M. Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA. Biotechniques 40, 649–657 (2006).

    CAS  Article  Google Scholar 

  40. 40

    Lewis, F. & Maughan, N.J. Extraction of Total RNA from Formalin-Fixed Paraffin-Embedded Tissue. in A-Z of quantitative PCR (ed. Bustin, S.A.) (IUL, La Jolla, California, 2004).

    Google Scholar 

  41. 41

    Lekanne Deprez, R.H., Fijnvandraat, A.C., Ruijter, J.M. & Moorman, A.F. Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions. Anal. Biochem. 307, 63–69 (2002).

    CAS  Article  Google Scholar 

  42. 42

    Stanley, K.K. & Szewczuk, E. Multiplexed tandem PCR: gene profiling from small amounts of RNA using SYBR Green detection. Nucleic Acids Res 33, e180 (2005).

    Article  Google Scholar 

  43. 43

    Hilscher, C., Vahrson, W. & Dittmer, D.P. Faster quantitative real-time PCR protocols may lose sensitivity and show increased variability. Nucleic Acids Res. 33, e182 (2005).

    Article  Google Scholar 

  44. 44

    Hartshorn, C., Anshelevich, A. & Wangh, L.J. Rapid, single-tube method for quantitative preparation and analysis of RNA and DNA in samples as small as one cell. BMC Biotechnol 5, 2 (2005).

    Article  Google Scholar 

  45. 45

    Pattyn, F., Speleman, F., De Paepe, A. & Vandesompele, J. RTPrimerDB: the real-time PCR primer and probe database. Nucleic Acids Res. 31, 122–123 (2003).

    CAS  Article  Google Scholar 

  46. 46

    Pattyn, F., Robbrecht, P., De Paepe, A., Speleman, F. & Vandesompele, J. RTPrimerDB: the real-time PCR primer and probe database, major update 2006. Nucleic Acids Res. 34, 684–688 (2006).

    Article  Google Scholar 

  47. 47

    Wang, X. & Seed, B. A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res. 31, 154 (2003).

    Article  Google Scholar 

  48. 48

    Reynisson, E., Josefsen, M.H., Krause, M. & Hoorfar, J. Evaluation of probe chemistries and platforms to improve the detection limit of real-time PCR. J. Microbiol. Methods 66, 206–216 (2005).

    Article  Google Scholar 

  49. 49

    Huang, Z., Fasco, M.J. & Kaminsky, L.S. Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR. Biotechniques 20, 1012–1020 (1996).

    CAS  Article  Google Scholar 

  50. 50

    Hengen, P.N. Is RNase-free really RNase for free? 21, 112–113 (1996).

  51. 51

    Imbeaud, S. et al. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res. 33, 56 (2005).

    Article  Google Scholar 

  52. 52

    Sambrook, J., MacCallum, P. & Russell, D. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2001).

    Google Scholar 

  53. 53

    Huggett, J., Dheda, K., Bustin, S. & Zumla, A. Real-time RT-PCR normalization; strategies and considerations. Genes Immun. 6, 279–284 (2005).

    CAS  Article  Google Scholar 

  54. 54

    Goossens, K. et al. Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos. BMC Dev. Biol. 5, 27 (2005).

    Article  Google Scholar 

  55. 55

    Tricarico, C. et al. Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. Anal.Biochem. 309, 293–300 (2002).

    CAS  Article  Google Scholar 

  56. 56

    Dheda, K. et al. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Anal. Biochem. 344, 141–143 (2005).

    CAS  Article  Google Scholar 

  57. 57

    Perez-Novo, C.A. et al. Impact of RNA quality on reference gene expression stability. Biotechniques 39 52, 54, 56 (2005).

    CAS  Article  Google Scholar 

  58. 58

    Vandesompele, J. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, 0034.1–0034.11 (2002).

    Article  Google Scholar 

  59. 59

    Pfaffl, M.W., Tichopad, A., Prgomet, C. & Neuvians, T.P. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26, 509–515 (2004).

    CAS  Article  Google Scholar 

  60. 60

    Kanno, J. et al. “Per cell” normalization method for mRNA measurement by quantitative PCR and microarrays. BMC Genomics 7, 64 (2006).

    Article  Google Scholar 

  61. 61

    Bauer, P., Rolfs, A., Regitz-Zagrosek, V., Hildebrandt, A. & Fleck, E. Use of manganese in RT-PCR eliminates PCR artifacts resulting from DNase I digestion. Biotechniques 22, 1128–1132 (1997).

    CAS  Article  Google Scholar 

  62. 62

    Nemeth, E. et al. IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J. Clin. Invest. 113, 1271–1276 (2004).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We acknowledge numerous fruitful discussions with J. Huggett, M. Kubista, R. Mueller, S. Mueller, M. Pfaffl, G. Shipley and J. Vandesompele. We are grateful to N. Gerke (Eppendorf AG) and J. Stolte (EMBL Heidelberg) for the experiment described in Figure 4, which was conducted at the 3rd EMBO RT-qPCR workshop at EMBL Heidelberg, 2006.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Stephen A Bustin.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Nolan, T., Hands, R. & Bustin, S. Quantification of mRNA using real-time RT-PCR. Nat Protoc 1, 1559–1582 (2006). https://doi.org/10.1038/nprot.2006.236

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing